1. Field of the Invention
The present invention relates to a use of NRP1 gene as a target for the development of an asthma marker or a therapeutic agent for asthma.
2. Description of the Related Art
Anaphylaxis, allergic rhinitis, asthma, atopic dermatitis, and urticaria are the examples of allergic diseases whose incidence rates have been increased world-widely (Wuthrich B. Int. Arch. Allergy Appl. Immunol., 90, pp 3-10, 1989). Among the said allergic diseases, asthma is characterized by bronchial hyperresponsiveness, which leads to chronic airway inflammation with carrying such symptoms as wheezing, dyspnea, and cough which are caused by extensive narrowness of the airway. Asthma can be reversed or improved naturally or by treatment (Minoguchi K and Adachi M. Pathophysiology of asthma. In: Chemiack N S, Altose M D, Homma I, editors. Rehabilitation of the patient with respiratory disease. New York: McGraw-Hill, pp 97-104, 1999).
Asthma is generally recognized as chronic inflammatory disease which is caused by the migration and infiltration of inflammatory cells proliferated, differentiated, and activated by interleukin-4, 5, and 13 generated by TH2 immunocytes into and around the airway (Elias J A, et al., J. Clin. Invest., 111, pp 291-297, 2003). At this time, the activated inflammatory cells such as eosinophils, mast cells and alveolar macrophages secret various inflammation mediators (cysteinyl leukotriene, prostaglandin, etc), which play a critical role in bronchoconstriction (Maggi E., Immunotechnology, 3, pp 233-244, 1998; Pawankar R., Curr. Opin. Allergy Clin. Immunol., 1, pp 3-6, 2001; Barnes P J, et al., Pharmacol Rev., 50, pp 515-596, 1998).
Productions of cytokines involved in the activation of inflammatory cells such as IL-4, IL-5, and IL-13, and immunoglobulin E, as well as biosynthesis of cysteinyl leukotriene secreted from inflammatory cells such as eosinophils mediated by the said cytokines and immunoglobulin E are regarded as major reasons of inflammation and allergic reaction and further asthma caused by such inflammation and allergic reaction. Therefore, studies are actively undergoing to develop drugs to inhibit the productions of the said cytokines, immunoglobulin E, and cysteinyl leukotriene. In particular, to understand on the allergic disease including asthma and rhino conjunctivitis, animal models have been used for the study. Animal models not only help the understanding of immunological mechanism in relation to allergic reaction but also enable the evaluations of novel drugs under development.
Neuropilin (NRP) consists of Neuropilin 1 and Neuropilin 2. Neuropilin 1 and Neuropilin 2 were first found in neurons (Takagi S et al., Dev Biol, 1987). Since then, they were identified as semaphorin 3A (SEMA3A) receptors mediating axon guidance with inhibiting the expression not in axons but in Growth cones (He Z, Cell, 1997). And then, the role of NRP as vascular endothelial growth factor (VEGF) family receptor known to be involved in angiogenesis receptor has been disclosed (Soker S et al., Cell, 1998).
NRP is a single transmembranal glycoprotein having the molecular weight of 130˜140 kDa. NRP1 is composed of approximately 923 amino acids and NRP2 is composed of approximately 926 amino acids. They both have similar domain structure with overall 44% amino acid homology (Caroline PELLET-MANY et al., Biochem J., 2008).
NRP1 and NRP2 are expressed in a variety of human tumor cell lines and human neoplasms (Bielenberg, D. R et al, Exp. Cell Res, 2006; Soker, S. et al, Cell, 1998; Ellis, L. M., Cancer Ther, 2006). It has been known that they are involved in the activities of VEGF and semaphorin affecting proliferation, survival, and migration of cancer cells (Bachelder, R. E. et al, Cancer Res, 2001; Chabbert-de Ponnat et al, J. Invest. Dermatol., 2006; Miao et al, FASEB J., 2000). NRP1 is found in the samples of patients with colon cancer, breast cancer, lung cancer, prostatic cancer, and pancreatic cancer, but not found in normal epithelium tissues. NRP1 is also found in various tumors such as neuroblastoma, melanoma, and astrocytoma. NRP2 is found in lung cancer, neuroblastoma, ostersarcoma, pancreatic cancer, and bladder cancer, according to previous reports. NRP1 is also expressed in a variety of cells including bone marrow derived progenitor cells, bovine granulosa platelets, granulosa cells, and theca cells, etc (Caroline PELLET-MANY et al, Biochem J., 2008). NRP1 is expressed in naïve T-cells and immature antigen-presenting cells, and is essential to induce the proliferation and differentiation of mature T-cells reacting with antigen-presenting cells mediating antigen elimination after the first immune response in the secondary lymphoid organs (Matthies, A. M., et al, Am. J. Pathol., 2002).
According to other reports, NRP1 is over-expressed by tissue wound healing reaction, suggesting that NRP1 is involved in regeneration and recovery. When optic nerve is damaged and needs to be regenerated in Xenopus, NRP1 expression is increased and even after healing, NRP1 up-regulation continues for further several weeks (Matthies, A. M., et al, Am. J. Pathol., 2002). NRP1 is found in a large scale in wound angiogenesis. Wound angiogenesis is reduced by anti-NRP1 antibody, suggesting that NRP1 plays an important role in angiogenesis (Elena Gerettil et al, Cell Adhesion & Migration, 2007).
To disclose the functions of NRP, numbers of animal models have been used. According to the primary report saying that NRP1 was involved in angiogenesis, embryo of the transgenic mouse was in danger by NRP1 over-expression with showing such symptoms as excessive angiogenesis, expanded blood vessels, bleeding, and abnormal heart and limbs development (Herzog Y et al., Mech Dev., 2001). In the NRP1 knock-out mouse, embryo was deceased in uterus on the 12.5th and 13.5th day from the development with showing abnormal yorksac, unusual nerve angiogenesis, blood vessel system disorder, branching deficiency, capillary network deficiency, immature bronchial arches, abnormal dorsal aorta, and cardiovascular disorder including aortic arches (Yuan L et al, Development, 2002). In the NRP2 knock-out mouse, artery and vein were developed normally, but lymph node and microvessel formation was significantly decreased (Yuan L et al, Development, 2002). In the NRP1/NRP2 knock-out mouse, embryo was deceased on the 8.5th day from the development with showing avascular yorksac, growth suppression, and more serious vascular disorder such as vascular development deficiency, capillary formation deficiency, and branching deficiency (Eichmann A et al, Int J Dev Biol, 2005).
Matrix metallopeptidase 9 (MMP-9) is involved in outer cell wall matrix degradation not only in normal physiological processes including embryo development, regeneration, and tissue remodeling, etc, not also in disease processes such as arthritis and metastasis. Most MMPs are secreted in the form of inactive proproteins, which are then divided by extracellular proteinases. The enzyme encoded by MMP-9 decomposes collagen IV and V. According to the previous studies using rhesus monkey, the enzyme encoded by MMP-9 was involved in the mobility of hematopoietic progenitor cells originated from bone marrow mediated by IL-8. According to other studies using animals of the rat genus, MMP-9 was involved in tissue remodeling in relation to tumor.
MMP-9 is generated by a specific stimulus in macrophages (Welgus H G et al, J Clin Invest, 1990), eosinophils (Hno I et al, Am J Respir Cell Mol. Biol., 1997), macrophages (Kanbe N, et al., Eur J Immunol, 1999), and dendritic cells (Bartholome E J, J Interferon Cytokine Res, 2001). MMP-9 is a kind of inflammatory molecule that plays a role in maintaining immune response (Renckens R. et al, J Immunol, 2006) and at the same time is involved in the regeneration of damaged tissues. MMP-9 is directly involved in airway inflammation, so MMP-9 expression is increased in sputum. MMP-9 is functioning to make airway thicker (Matsumoto H, et al, Thorax, 2005). There have been numbers of reports saying that MMP-9 is involved in respiratory disease such as asthma, but the exact mechanism thereof has not been disclosed, yet.
The preset inventors have been studied for the development of a novel asthma marker or a therapeutic agent for asthma. As a result, the inventors completed this invention by confirming the followings: Expressions of inflammatory cytokine and NRP1 were increased in cells when D. pteronissinus extract known to cause asthma was treated to the cells; NRP1 expression was significantly increased in peripheral blood cells of asthma patient, compared with that in the normal control; MMP-9 mRNA, protein activity, and enzyme activity were all decreased by NRP1 expression or activity inhibitor; and MMP-9 promoter activity was reduced by NRP1 expression or activity inhibitor dose-dependently; which all suggested that NRP1 (neuropilin 1) gene could be effectively used as a marker for the diagnosis of asthma and thereby the said gene marker could be effectively used for the screening of a therapeutic agent for asthma.